The overall goal of this proposed Phase I project is to determine scientific merit and feasibility of constructing highly specific recombinant single chain variable fragment (scFv) antibodies to Clostridium difficile toxin A and toxin B, and detect their presence for rapid, inexpensive, and sensitive diagnosis of Clostridium difficile associated diarrhea (CDAD). We propose to develop recombinant C. difficile anti-toxin A and anti-toxin B scFv and determine their utility as vital tools for the next generation of CDAD diagnostic products. Specific aims are to: (1) evaluate recombinant C. difficile anti-toxin B scFv for ability to detect toxin B, (2) develop a panel of recombinant C. difficile anti-toxin A scFv using an anti-toxin A monoclonal antibody as starting material, using mutagenesis and phage display to improve scFv affinity, (3) evaluate recombinant C. difficile anti-toxin A scFv for ability to detect toxin A, and (4) develop platform technology that demonstrates binding of anti-toxin A and anti-toxin B scFv for use in clinical laboratories for diagnosis of CDAD. ScFv will be evaluated in lateral flow assay and ELISA formats (as both detection and capture antibodies), and fluorescence polarization platforms. Successful completion of the Aims will allow detecting toxin A and toxin B in clinical laboratories for detection of CDAD. Our long term goal is to develop (Phase II study) and market (Phase III commercialization) a superior test for CDAD that meets or exceeds the sensitivities and specificities of currently available tests, is incomparable because it features rapid turn around time and minimizes human and economic loss.